Chronic inflammation contributes to atherosclerosis and evidence suggests that innate immune defense mechanisms may interact with proinflammatory pathways and lead to the development of arterial plaques. The innate immune system allows cells to distinguish between pathogen and self by utilizing the signals from Toll-like receptors (TLRs). Recent information also directly implicates signaling by the TLR pathway in these processes, establishing a link between atherosclerosis, defense against foreign pathogens, and endogenously generated inflammatory ligands. TLRs are expressed in vascular cells, specifically atherosclerotic plaque and monocytes. Recent major studies also support the concept that acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets are central in acute thrombotic syndromes and accumulating data demonstrate their role in inflammation, TLRs have not been directly implicated in platelet function. In preliminary data utilizing microarray analyses, we found distinct patterns of platelet gene expression, specifically, TLR2 was increased in patients with acute coronary events and increased TLR1 was associated the presence of diabetes. These findings were confirmed by quantitative RT-PCR, flow cytometry, and confocal microscopy. Proteomic analysis demonstrated the presence of TLR2 in platelets and identified specific TLR2-interacting proteins. Most importantly, incubation of platelets with a TLR2 ligand dose-dependently induced platelet activation and aggregation. Thus, the central hypothesis of this proposal is that innate immunity is relevant to the thrombo-inflammatory response and it is mediated by the expression of TLRs on platelets. To investigate this hypothesis, we propose: Specific aim 1. To further characterize platelet TLR1/TLR2 and determine its role in platelet function. Specific aim 2. To identify and characterize downstream TLR2-dependent signaling pathways in platelets. Specific aim 3. To determine the role of platelet TLR2 in the formation of thrombosis in vivo using a murine model of TLR2 deficiency. Specific aim 4. To study how TLR2 mediates the effects of obesity induced inflammation and platelet reactivity in vivo using a murine model of diet induced obesity. In summary, these studies will further define the role of innate immunity in the processes regulating thrombo-inflammation. These studies will specifically explore the contribution of platelet TLR2 to platelet function and thrombosis in the setting of inflammation.